This is a proposal to implement a novel high throughput assay system to detect inhibitors of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) formation and/or maintenance. HBV cccDNA is essential to the virus life cycle and its elimination during chronic infection is considered critical to durable therapy. However, because of the limitations of current HBV tissue culture systems, including the impracticality of detecting cccDNA itself, cccDNA has not been rigorously targeted in high throughput screening of small molecule libraries. In this proposal, a novel tissue culture line that expresses hepatitisB e antigen (HBeAg) in a cccDNA-dependent manner will be used to screen a library of extracts produced from natural biological sources. This cell line produces viral pregenome transcripts from a stably integrated, linear, inducible HBV genome (transgene), leading to replication of full-length viral genome and cccDNA formation; subsequently, intact HBeAg RNA and protein are only made from transcripts produced from the cccDNA template. In an HTS campaign, compounds that lower HBeAg, as detected by an immunological assay, would be considered candidate inhibitors of cccDNA formation, expression or longevity. Thus far, this system has been used to screen a library of ~85,000 synthetic small molecule compounds, resulting in the identification of two compounds that repress cccDNA through inhibition of a specific step in its formation, constituting a unique mechanism of action. While these compounds are currently under study, we propose to extend our efforts to a unique library of 80,000 extracts produced from approximately 36,000 microbial and 9,000 plant species. The botanical coverage alone includes over 60% of all known plant genera. This library was assembled and used by Merck and Co. as the starting material for several marketed drugs, and was until recently completely proprietary and inaccessible. Through collaboration with the Natural Products Discovery Institute, we will screen this unique resource for naturally-derived compounds with anti-cccDNA activity. The extracts scored as hits will be confirmed, and the active compounds will identified through fractionation and our medicinal chemistry expertise. Thus, we will expand the pool of compounds to be used for HBV research, or even the possible derivation of transformational therapies for chronic hepatitis B.